[unreadable] As the prevalence of Diabetes Mellitus increases worldwide, the need for recognizing the major cellular and molecular processes that underlie progression of the disease becomes more urgent. It is now clear that at the time or shortly after onset of "beta cell dysfunction" in diabetes, beta cells develop "secretory pathway stress" initiated within a compartment in the cell called the endoplasmic reticulum (ER). ER stress includes misfolding of proinsulin, the beta cell's major secretory protein product - the precursor in insulin biosynthesis. I propose that proinsulin misfolding causes further beta cell dysfunction and demise, ultimately decreasing pancreatic insulin mass. Indeed in the Akita mouse, a point mutation in just one genetic copy of proinsulin-ll (with two normal copies of proinsulin-l and one normal proinsulin-ll) causes sufficient misfolded proinsulin to produce diabetes in all animals with the mutation (so-called "dominant negative" behavior). I have been interested to know whether misfolded proinsulin can attack other proinsulin molecules in the ER and confer upon them the mutant behavior, thereby leading to cell toxicity and loss of beta cell (and insulin) mass. However, conventional methodologies cannot distinguish normal proinsulin from misfolded proinsulin. In this application, I have developed a novel approach to model the folding of normal proinsulin and to examine possible interactions with misfolded proinsulin by generating a transgenic mouse expressing (selectively in (-cells) a human proinsulin fusion protein containing enhanced green fluorescent protein within the C-peptide (midregion) of the proinsulin molecule (called hProins-CpepGFP). Preliminary studies are presented, indicating that this fusion protein exhibits the typical features of normal proinsulin folding, trafficking, processing, and secretion. In this grant application I intend to breed this transgenic mouse with Akita mice or, alternatively, to introduce the Akita mutation into the hProins-CpepGFP construct, in order to directly examine interactions of hProins-CpepGFP with misfolded proinsulin. Using these models, (-cell (tagged) insulin mass can be followed. [unreadable] A combination of biochemical and morphological approaches are proposed to understand the relationship of proinsulin misfolding to progression of diabetes. The model presented opens new avenues to the development and testing of potential therapeutic interventions that may ultimately help preserve insulin secretory function in humans with diabetes mellitus. [unreadable] [unreadable]